Enzymes of the heme biosynthesis pathway, which are essential for all life and have been conserved throughout evolution, are associated with human diseases and offer potential as targets for antibiotics, fungicides, and herbicides. During the initial funding period we determined four new protein structures and performed associated mutagenic and biochemical analyses. The current proposal emphasizes determination of substrate/product complex structures, with focus on U3S (4th enzyme of heme biosynthesis pathway), URO-D (5th enzyme), PROTO-OX (7th enzyme), and Met8p (branch pathway of siroheme biosynthesis).We have recently co-crystallized URO-D in complex with product by use of a coupled enzyme system that circumvents the difficulty of extreme oxygen sensitivity of pathway intermediates. Preliminary data collected to 2.2A resolution reveal that the product is well ordered in the active site and indicates the location of the catalytic center. Remarkably, an invariant aspartate side chain coordinates all four of the tetrapyrrole amides and may serve as a pivot about which the substrate rotates to allow sequential decarboxylation of all four acetate side chains. Experiments are proposed to test questions of mechanism, including the importance of specific residues for binding and catalysis, the importance of a loop that covers the active site, and the functional significance of dimerization. The approach developed for the URO-D/product complex will be adapted to obtain complexes of inactive URO-D mutants with substrate. This approach will also be adapted to obtain substrate and product complex structures of U3S and Met8p, for which we have already determined the apo structures. Finally, the PROTO-OX structure, towards which we have already collected 2.3A native data, will be of special interest for the development of novel inhibitors.